Cryptococcus neoformans is a major pathogen in AIDS patients causing life-threatening meningoencephalitis in a large number of HIV+ individuals. Furthermore, groups classically at risk for cryptococcosis, the elderly, transplant recipients and those immunosuppressed with steroids or chemotherapy, are continually rising. Previous studies by us and others have shown a strong association between laccase-dependent melanin production by C. neoformans in vitro and virulence of the organism in experimental animals. We have shown that laccase is a cell wall-associated virulence factor by multiple lines of evidence: 1) localization by immuno-electron microscopy using an anti-laccase monoclonal antibody, 2) localization to the periphery of a green-fluorescent protein-tagged recombinant laccase by confocal microscopy, 3) localization of enzyme activity and laccase immunoreactive protein to cell wall preparations of C. neoformans. We have also developed a method of insertional mutagenesis and have isolated numerous laccase-deficient mutants. The overall objective of this proposal is to test the hypothesis that accurate cell wall targeting of laccase is required for the virulence of Cryptococcus neoformans. Detailed understanding of virulence-related trafficking may allow the identification of other novel virulence factors which share these same pathways and may allow the identification of unique trafficking inhibitors that disrupt the function of multiple virulence factors with a single agent. In Specific Aim 1, we will identify targeting-specific amino acid sequences in laccase. We will use confocal microscopy to compare the cellular localization of expressed green fluorescent protein-tagged laccase in C. neoformans with an identical fusion protein containing deletions of the putative N-terminal vacuolar targeting sequence E25SPT or C-terminal clathrin-coated pit sequence Y426GFNNI. Additional deletions will be performed within the laccase N-terminus and C-terminus of the fusion protein to determine if other targeting sequences may be present. In Specific Aim 2, we will identify genes and inhibitors involved in laccase trafficking by studying laccase trafficking after mutation of sentinel genes involved in actin-dependent, clathrin-dependent and vacuolar-dependent clathrin-independent trafficking pathways. In Specific Aim 3 we will identify genes involved in laccase trafficking by studying laccase-deficient insertional mutants which show secretory defects in laccase secretion. Each of the mutants identified in Specific Aims 2 and 3 will then be assessed for production of other virulence-related factors such as capsule, urease, phospholipase and virulence in a mouse model.